Examining the Role of RNA Secondary Structure on Human RNA Polymerase II Pausing

The process of transcription, in which DNA is converted to RNA, is an integral process in the expression of our genetic information. Regulation of transcription is critical for healthy, functioning cells and defects in transcriptional regulation can cause a plethora of ill health effects including cancer and improper cardiac or neuronal development. Human RNA Polymerase II (Pol II) is the enzyme that transcribes all protein-coding and most non-coding RNA genes in the human genome. Within the past 10 years, it has become evident that Pol II function is regulated after it initiates transcription; specifically, the Pol II enzyme pauses after transcribing 20-100 bases of DNA into RNA. This process is highly regulated by numerous protein factors, but no groups have yet addressed the nascent RNA present during transcription as a potential regulatory factor. This project examines the nascent RNA secondary structure and its potential for regulating Pol II pausing at the native HSP70 human promoter. A combination of approaches was used to dissect the importance of RNA secondary structure in Pol II pausing. First, in vitro transcription assays were used to test the role of RNA secondary structure in pausing by incorporating structure disrupting nucleotide triphosphate analogues into the nascent RNA. Second, SHAPE chemical probing and structural prediction algorithms were used to predict the secondary structure of the HSP70 nascent RNA. By utilizing both in vitro chemical methods and directed structural prediction, this project has begun to dissect the role of RNA secondary structure in human Pol II pausing